Luminescent analyses with enhanced storage stability

Abstract

The storage stability of peroxidase-reactive luminescent cocktails which employ chemiluminescent 2,3-dihydro-1,4-phthalazinediones, oxidants reactant therewith to cause light emission in peroxidase-mediated luminescent reactions, and phenolic sensitivity enhancers is improved by maintaining the pH of the cocktail prior to use at a value in the range of about 3 to about 6 and preferably at a pH of about 5. An analyte sample may be subjected to a reaction resulting in a peroxidase-containing phase, the peroxidase content of which is related to the quantity of analyte in the sample, and the peroxidase phase may then be combined with the thus-described cocktail and an aqueous alkaline organic buffer to raise the pH of the resulting reaction mixture to a value in the range of 7-9, favoring light emission.

What I claimed is: 
     
       1. A storage-stable reagent useful in luminescence-monitored enzyme assays and which is capable, when reacted with peroxidase, in an alkaline environment, of undergoing a luminescent reaction, the reagent comprising (a) a chemiluminescent reagent and an oxidant reactive therewith to cause light emission in a peroxidase-mediated luminescent reaction,   (b) a sensitivity enhancer capable of increasing the signal-to-background ratio of said luminescent reaction, and   (c) a pH-regulating composition regulating the pH of the reagent to a value in the range of about 3 to about 6.   
     
     
       2. The reagent of claim 1 in which the chemiluminescent material is a 2,3-dihydro-1,4phthalazinedione and the phenolic sensitivity enhancer is 4-iodophenol or 4-phenylphenol. 
     
     
       3. The reagent of claim 2 in which the oxidant is hydrogen peroxide, perborate ion or urea peroxide. 
     
     
       4. The reagent of claim 1 in which the pH-regulating composition is an acetate or citrate buffer. 
     
     
       5. The reagent of claim 1 in which the pH of the reagent is about 5.0. 
     
     
       6. The reagent of claim 1 including calcium chloride in an amount sufficient to substantially increase peroxidase-mediated luminescence output without significantly increasing background luminescence output. 
     
     
       7. A storage-stable peroxidase-reactive reagent containing (a) a 2,3-dihydro-1,4-phthalazinedione and an oxidant reactive therewith to cause light emission in a peroxidase-mediated luminescent reaction in an alkaline environment,   (b) a phenolic sensitivity enhancer, and   (c) a pH-regulating composition regulating the pH of the reagent to a value in the range of about 3 to about 6.   
     
     
       8. The reagent of claim 7 wherein the pH-regulating composition regulates the pH of the reagent to about 5.0. 
     
     
       9. An assay kit for performing a peroxidase-mediated, luminescence-monitored assay for an analyte, comprising (a) a vessel containing a peroxidase-reactive reagent comprising (i) a chemiluminescent 2,3-dihydro-1,4-phthalazinedione and an oxidant reactive therewith to cause light emission in a peroxidase-mediated luminescent reaction in an alkaline environment,   (ii) a phenolic sensitivity enhancer, and   (iii) a buffer controlling the pH of the reagent to a value in the range of about 3 to about 6; and     (b) a separate vessel containing an organic alkaline aqueous buffer capable of adjusting the pH of the first reagent to an alkaline pH favoring light emission.   
     
     
       10. The assay kit of claim 9 in which the organic alkaline aqueous buffer is further characterized by producing negligible buffer-mediated luminescence. 
     
     
       11. The assay kit of claim 10 in which the alkaline buffer is Tricine or glycylglycine. 
     
     
       12. The assay kit of claim 10 in which the peroxidase-reactive reagent includes an acetate or citrate buffer maintaining the pH of that reagent at about 5.0. 
     
     
       13. The kit of claim 9 including, in the peroxidase-reactive reagent, the alkaline buffer or both, CaCl 2  in an amount sufficient to substantially increase peroxidase-mediated luminescence output without significantly increasing background luminescence output. 
     
     
       14. The assay kit of claim 9 including a peroxidase conjugate of said analyte or of a binding partner to the analyte. 
     
     
       15. An assay kit for performing a peroxidase-mediated, luminescence-monitored assay for an analyte, comprising (a) a vessel containing a peroxidase-reactive reagent including a chemiluminescent composition containing luminol or isoluminol, an oxidant which is hydrogen peroxide, perborate ion or urea peroxide, a sensitivity enhancer which is 4-iodophenol or 4-phenylphenol, and a buffer maintaining the pH of the reagent at a value in the range of about 3-6, and   (b) a separate vessel containing an organic buffer having a pK in the range of 7.0-9.5 and which includes Tricine or glycylglycine.   
     
     
       16. The kit of claim 15 including, in the alkaline buffer, sufficient CaCl 2  to substantially increase peroxidase-mediated luminescence output without significantly increasing background luminescence output. 
     
     
       17. The assay kit of claim 15 including a peroxidase conjugate of the analyte or of a binding partner to the analyte. 
     
     
       18. A method for performing an assay for an analyte in an analyte sample, in which the analyte sample is subjected to a reaction resulting in a peroxidase-containing phase, the quantity of peroxidase in which is related to the quantity of analyte in the sample, the method comprising: (a) combining said peroxidase-containing phase with (i) a peroxidase-reactive reagent comprising a chemiluminescent 2,3-dihydro -1,4-phthalazinedione, an oxidant reactive therewith to cause light emission in a peroxidase-mediated luminescent reaction in an alkaline environment, a phenolic sensitivity enhancer, and a buffer which maintains the pH of the reagent in the range of about 3 to about 6, and   (ii) an organic alkaline aqueous buffer to provide the resulting reaction mixture with an alkaline pH favoring light emission therefrom; and     (b) measuring the resulting emitted light.   
     
     
       19. The method of claim 18 in which the anic alkaline buffer is characterized by producing negligible buffer-mediated luminescence. 
     
     
       20. The method of claim 18 in which the peroxidase-reactive reagent is maintained, prior to use, at a pH of about 5.0. 
     
     
       21. The method of claim 18 in which organic alkaline aqueous buffer maintains the pH of the reaction mixture at a value in the range of 7-9. 
     
     
       22. The method of claim 18, in which the organic alkaline aqueous buffer includes Tricine or glycylglycine. 
     
     
       23. The method of claim 18 wherein CaCl 2 , in sufficient quantity to enhance the peroxidase-mediated luminescence output without significantly increasing background luminescence output, is combined with the chemiluminescent reagent. 
     
     
       24. A method for performing an assay for an analyte in an analyte sample, in which the analyte sample is subjected to a reaction resulting in a peroxidase-containing phase, the quantity of peroxidase in which phase is related to the quantity of analyte in the sample, the method comprising combining the peroxidase-containing phase with (a) a peroxidase-reactive reagent having a pH in the range of 3 to 6, comprising a chemiluminescent reagent and an oxidant reactive therewith to cause light emission in a peroxidase-mediated luminescent reaction in an alkaline environment, and a sensitivity enhancer capable of increasing the signal-to-background ratio of said luminescent reaction,   (b) an organic, alkaline buffer to provide the resulting reaction mixture with an alkaline pH favoring light emission, and   (c) measuring the resulting emitted light.   
     
     
       25. The method of claim 24 wherein the alkaline buffer is provided with sufficient CaCl 2  to enhance the peroxidase-mediated luminescence output without significantly increasing background luminescence output.